The molecular basis for stable variation and mutation in heteroploid mammalian cell lines is investigated. In the first group of studies, hypoxanthine phosphoribosyltransferase (HPRT) is used as a selectable marker to explore the mechanism of cellular mutation to drug resistance. Most animal cells exhibit differential sensitivity to the purine analogues 8-azaguanine and 6-thioguanine, and our studies indicate that this is due to intrinsic differences in the substrate binding affinity of the normal HPRT to these analogues. Mouse cell lines A9 and RAG, which are deficient in HPRT and resistant to 8-azaguanine and 6-thiogaunine, have been mutagenized to generate a series of HPRT-positive revertants. The revertant clones are being analyzed for evidence of a typical HPRT activity, by substrate specificity electrophoretic mobility, thermal stability and gene complementation in cell hybrids. We have also isolated mutagen-induced derivatives of A9 revertants, which are simultaneously resistant to 8-azaguanine and HAT but sensitive to 6-thioguanine. Further analysis of these mutants may provide new insights into the nature of somatic mutations to drug resistance in general and into the mechanism of differential cellular resistance to purine analogues in particular. In the second group of experiments, the role of somatic mutation in malignant transformation is studied by using athymic nude mice as a test system for cellular tumorigenicity. After a single-step mutagenesis, we have isolated human diploid cell mutants that are stably and specifically transformed with respect to anchorage regulation of cell growth. These anchorage-transformed cells are normal in morphology, chromosome constitution and susceptibility to senescence in vitro, but show high plating efficiency in semi-solid medium and absence of contact inhibition of growth. We are studying these mutants to determine if they are more susceptible to fully malignant transformation than the original parental cells. BIBLIOGRAPHIC REFERENCES: V.H. Freedman, A.L. Brown, H.P. Klinger and S. Shin. Mass production of animal cells in nude mice with retention of cell specific markers. Exp. Cell Res. (in press) (1976). T.F. Donahue, O.P. van Diggelen and S. Shin. Biochemical basis for differential cellular resistance to 8-azaguanine andd 6-thioguanine. J. Cell Biol. (in press) (1976).